This invention relates generally to the identification and characterization of fungal genes and proteins, and in particular to mitotic spindle proteins useful as development candidates for anti-mitotic agents and processes using such agents.
The mitotic spindle undergoes a remarkable series of transitions in response to cell cycle control signals. At each mitotic cell division, the spindle assembles, it forms attachments to each chromosome, it orients itself properly within the cell, and then, with extraordinarily high fidelity, it carries out chromosome segregation. Then it disassembles.
Proper spindle assembly and function involves coordination of many events and processes including modulation of microtubule dynamics and creation of at least three distinct microtubule populations (kinetochore, polar, and astral microtubules). In addition, connections must be established between different spindle microtubule subpopulations, between spindle microtubules and chromosomes, between spindle microtubules and microtubule-associated proteins and motor proteins, and between spindle microtubules and the cell cortex (reviewed by Waters and Salmon, 1997). Proper spindle assembly is monitored by a cellular surveillance system which activates a mitotic checkpoint if the spindle is not assembled correctly (reviewed by Hardwick, 1998; Rudner and Murray, 1996). Once the spindle is assembled, a carefully orchestrated set of molecular events results in chromosome to pole movement (anaphase A) and separation of spindle poles (anaphase B).
Genetic approaches to the study of spindle mechanics and regulation in S. cerevisiae, S. pombe, A. nidulans, and in a variety of other organisms have complemented studies in Xenopus extracts and mammalian and plant cells (reviewed by Nicklas, 1997; Sobel, 1997). Each different approach has provided an extremely powerful and unique avenue toward identification of mitotic spindle components and elucidation of their functions. Budding yeast contains five kinesin-related motor proteins and one dynein (reviewed by Winsor and Schiebel, 1997). Elegant genetic studies in yeast have revealed how the forces generated by these proteins work both synergistically and antagonistically to assemble and orient spindles, and to separate chromosomes (Cottingham and Hoyt, 1997; Gambino et al., 1984; Oakley and Morris, 1980; Oakley and Rinehart, 1985; Saunders and Hoyt, 1992).
It is believed that a large number of proteins in the spindle function in concert with tubulin, the major spindle protein. Genetic studies have identified and provided functional tests of .gamma.-tubulin and many other proteins associated with spindle pole bodies (Marschall et al., 1996; Oakley, 1994; Rout and Kilmartin, 1990; Sobel and Snyder, 1995; Spang et al., 1995). Also, a number of spindle accessory proteins have been found and studied functionally (Berlin et al., 1990; Machin et al., 1995; Pasqualone and Huffaker, 1994; Pellman et al., 1995; Wang and Huffaker, 1997). These genetic studies have been particularly valuable both because non-tubulin spindle components are typically low in abundance, making their discovery difficult by other means, and because genetic analysis facilitates tests of function in vivo.
As indicated above, the mitotic spindle has been the subject of considerable research. The study of mitotic spindle proteins has yielded anti-mitotic compounds with important applications in cancer chemotherapy, and therapeutic agents targeted against fungal pathogens. For example, several plant and fungal secondary metabolites such as colchicine, vinblastine and taxol have been shown to interfere with mitotic spindle function in a wide variety of eukaryotes.
The demonstrated effectiveness of these anti-mitotic compounds in important medical and agricultural applications demonstrates the desirability of identifying and characterizing anti-mitotic compound development candidates.